WO2011098805A1 - Composés à base de sangliféhrine - Google Patents

Composés à base de sangliféhrine Download PDF

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Publication number
WO2011098805A1
WO2011098805A1 PCT/GB2011/050232 GB2011050232W WO2011098805A1 WO 2011098805 A1 WO2011098805 A1 WO 2011098805A1 GB 2011050232 W GB2011050232 W GB 2011050232W WO 2011098805 A1 WO2011098805 A1 WO 2011098805A1
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Prior art keywords
bond
compound according
aryl
sanglifehrin
alkyl
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PCT/GB2011/050232
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English (en)
Inventor
Steven James Moss
Matthew Alan Gregory
Barrie Wilkinson
Christine Janet Martin
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Biotica Technology Limited
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Priority claimed from GBGB1002094.9A external-priority patent/GB201002094D0/en
Priority claimed from GBGB1006124.0A external-priority patent/GB201006124D0/en
Application filed by Biotica Technology Limited filed Critical Biotica Technology Limited
Publication of WO2011098805A1 publication Critical patent/WO2011098805A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/04—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having less than three double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to sanglifehrin analogues, that are useful as cyclophilin inhibitors, e.g. in the treatment of viral infection especially infection by RNA viruses such as Hepatitis C virus (HCV) and Human Immunodeficiency Virus (HIV) and/or as
  • immunosuppressants e.g. for use in prophylaxis of transplant rejection and as anti-inflammatory agents, e.g. for use in treatment of inflammatory disorders.
  • the present invention also provides methods for their use in medicine, in particular for the treatment of HCV infection and for use as an immunosuppressant or anti-inflammatory agent, and in diseases where inhibition of the Mitochondrial Permeability Transition Pore (mPTP) is useful such as muscular dystrophy.
  • mPTP Mitochondrial Permeability Transition Pore
  • Hepatitis C virus is a positive strand RNA virus, and infection is a leading cause of post-transfusional hepatitis.
  • HCV is the most common chronic blood borne infection, and the leading cause of death from liver disease in United States.
  • the World Health Organization estimates that there are more than 170 million chronic carriers of HCV infection, which is about 3% of the world population.
  • the un-treated HCV-infected patients about 70%-85% develop chronic HCV infection, and are therefore at high risk to develop liver cirrhosis and hepatocellular carcinoma.
  • 50-76% of all cases of liver cancer and two- thirds of all liver transplants are due to chronic HCV infection (Manns et al, 2007).
  • HCV has a short life cycle and therefore development of drug resistance during drug therapy is common.
  • Novel, specifically targeted antiviral therapy for hepatitis C (STAT-C) drugs are being developed that target viral proteins such as viral RNA polymerase NS5B or viral protease NS3 (Jacobson et al, 2007; Parfieniuk et al., 2007).
  • novel compounds also are being developed that target human proteins (e.g. cyclophilins) rather than viral targets, which might be expected to lead to a reduction in incidence of resistance during drug therapy (Manns et al., 2007; Pockros, 2008; Pawlotsky J-M, 2005).
  • Cyclophilins are a family of cellular proteins that display peptidyl-prolyl cis-trans isomerase activity facilitating protein conformation changes and folding. CyPs are involved in cellular processes such as transcriptional regulation, immune response, protein secretion, and mitochondrial function. HCV virus recruits CyPs for its life cycle during human infection.
  • CyPs stimulate the RNA binding activity of the HCV non-structural protein NS5B RNA polymerase that promotes RNA replication, although several alternative hypotheses have been proposed including a requirement for CyP PPIase activity.
  • the ability to generate knockouts in mice Coldgan et al., 2000
  • human T cells Braaten and Luban, 2001 ) indicates that CyPA is optional for cell growth and survival..
  • Cyclosporine A (Inoue et al. 2003) ("CsA") and its closely structurally related non- immunosuppressive clinical analogues DEBIO-025 (Paeshuyse et al. 2006; Flisiak et al. 2008), NIM81 1 (Mathy et al. 2008) and SCY-635 (Hopkins et al., 2009) are known to bind to cyclophilins, and as cyclophilin inhibitors have shown in vitro and clinical efficacy in the treatment of HCV infection (Crabbe et al., 2009; Flisiak et al. 2008; Mathy et al.
  • DEBIO-025 the most clinically advanced cyclophilin inhibitor for the treatment of HCV, has shown in vitro and in vivo potency against the four most prevalent HCV genotypes (genotypes 1 , 2, 3, and 4). Resistance studies showed that mutations conferring resistance to DEBIO-025 were different from those reported for polymerase and protease inhibitors, and that there was no cross resistance with STAT-C resistant viral replicons. More importantly, DEBIO-025 also prevented the development of escape mutations that confer resistance to both protease and polymerase inhibitors (Crabbe et al., 2009).
  • CsA-based cyclophilin inhibitors in clinical development have a number of issues, which are thought to be related to their shared structural class, including: certain adverse events that can lead to a withdrawal of therapy and have limited the clinical dose levels; variable pharmacokinetics that can lead to variable efficacy; and an increased risk of drug-drug interactions that can lead to dosing issues.
  • DEBIO-025 and cyclosporine A are known to be inhibitors of biliary transporters such as bile salt export pumps and other hepatic transporters (especially MRP2/cMOAT/ABCC2) (Crabbe et al., 2009). It has been suggested that the interaction with biliary transporters, in particular MRP2, may be the cause of the hyperbilirubinaemia seen at high dose levels of DEBIO-025 (Nelson et al., 2009).
  • DEBIO-025 and cyclosporine A are substrates for metabolism by cytochrome P450 (especially CYP3A4), and are known to be substrates and inhibitors of human P- glycoprotein (MDR1 ) (Crabbe et al., 2009). Cyclosporine A has also been shown to be an inhibitor of CYP3A4 in vitro (Niwa et al., 2007). This indicates that there could be an increased risk of drug-drug interactions with other drugs that are CYP3A4 substrates, inducers or inhibitors such as for example ketoconazole, cimetidine and rifampicin. In addition, interactions are also expected with drugs that are subject to transport by P-glycoprotein (e.g.
  • CsA digoxin
  • CsA is also known to have highly variable pharmacokinetics, with early formulations showing oral bioavailability from 1 -89% (Kapurtzak et al., 2004). Without expensive monitoring of patient blood levels, this can lead to increased prevalence of side effects due to increased plasma levels, or reduced clinical response due to lowered plasma levels.
  • Sanglifehrins have also been shown to exhibit a lower immunosuppressive activity than CsA when tested in vitro (Sanglier et al., 1999; Fehr et al., 1999). SfA binds with high affinity to the CsA binding site of CyPA (Kallen et al., 2005;).
  • immunosuppressive agents with low toxicity for use in such areas as prophylaxis of transplant rejection, autoimmune, inflammatory and respiratory disorders, including Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease.
  • autoimmune inflammatory and respiratory disorders
  • Behcet syndrome uveitis
  • psoriasis atopic dermatitis
  • nephritic syndrome aplastic anaemia
  • biliary cirrhosis asthma
  • COPD chronic obstructive pulmonary disease
  • Sanglifehrins have been shown to have a novel mechanism of immunosuppressive activity (Zenke et al., 2001 ), and there is therefore an opportunity to develop agents with a mechanism of action different to current clinical agents, such as cyclosporine A, rapamycin and FK506.
  • Sanglifehrin A has been shown to be 10 fold less potent than Cyclosporine A, so the ideal novel agent would have improved potency and/or therapeutic window.
  • HIV Human Immunodeficiency Virus
  • Cyclophilin inhibitors such as CsA and DEBIO-025 have also shown potential utility in inhibition of HIV replication.
  • the cyclophilin inhibitors are thought to interfere with function of CyPA during progression/completion of HIV reverse transcription (Ptak et al., 2008).
  • DEBIO-025 only reduced HIV-1 RNA levels ⁇ 0.5 and >1 Iog10 copies/mL in nine and two patients respectively, whilst 27 of the treated patients showed no reduction in HIV-1 RNA levels (Steyn et al., 2006).
  • DEBIO-025 was trialled in HCV/HIV coinfected patients, and showed better efficacy against HCV, and the HIV clinical trials were discontinued (see Watashi et al., 2010).
  • MMT mitochondrial permeability transition
  • Cyclophilin D also known as Cyclophilin F
  • Cyclophilin D inhibitors may therefore be useful in indications where the mPTP opening has been implicated, such as muscular dystrophy, in particular Ullrich congenital muscular dystrophy and Bethlem myopathy (Millay et al., 2008, WO2008/084368, Palma et al., 2009), multiple sclerosis (Forte et al., 2009), diabetes (Fujimoto et al., 2010), amyotrophic lateral sclerosis (Martin 2009), bipolar disorder (Kubota et al., 2010), Alzheimer's disease (Du and Yan, 2010), Huntington's disease (Perry et al., 2010), recovery after myocardial infarction (Gomez et al., 2007) and chronic alchohol consumption (King
  • Cyclophilin inhibitors have potential activity against other viruses, such as Varicella- zoster virus (Ptak et al., 2008), Influenza A virus (Liu et al., 2009), Severe acute respiratory syndrome coronavirus and other human and feline coronaviruses (Chen et al., 2005, Ptak et al., 2008), Dengue virus (Kaul et al., 2009), Yellow fever virus (Qing et al., 2009), West Nile virus (Qing et al., 2009), Western equine encephalitis virus (Qing et al., 2009), Cytomegalovirus (Kawasaki et al., 2007) and Vaccinia virus (Castro et al., 2003).
  • viruses such as Varicella- zoster virus (Ptak et al., 2008), Influenza A virus (Liu et al., 2009), Severe acute respiratory syndrome coronavirus and other human and
  • such cyclophilin inhibitors have improved properties over the currently available cyclophilin inhibitors, including one or more of the following properties: improved water solubility, improved potency against HCV, reduced toxicity (including hepatotoxicity), improved pharmacological profile, such as high exposure to target organ (e.g.
  • the present invention discloses novel sanglifehrin analogues which may have one or more of the above properties.
  • novel ester derivatives which are anticipated to have improved potency against HCV, for example as shown by a low replicon EC50.
  • immunosuppressive agents which may have utility in the prophylaxis of transplant rejection, or in the treatment of autoimmune, inflammatory and respiratory disorders.
  • immunosuppressants have improved properties over the known sanglifehrins, including one or more of the following properties:
  • improved water solubility improved potency in immunosuppressive activity, such as might be seen in t-cell proliferation assays, reduced toxicity (including hepatotoxicity), improved pharmacological profile, such as high exposure to target organ and/or long half life (enabling less frequent dosing), reduced drug-drug interactions, such as via reduced levels of CYP3A4 metabolism and inhibition and reduced (Pgp) inhibition (enabling easier multi-drug
  • the present invention discloses novel sanglifehrin analogues which may have one or more of the above properties.
  • the present invention discloses novel ester derivatives, which have improved immunosuppressive potency, for example as shown by a low t-cell proliferation IC50.
  • the present invention provides novel macrocyclic sanglifehrin analogues, which have been generated by semisynthetic modification of native sanglifehrins. These analogues may be generated by dihydroxylation of a sanglifehrin, such as SfA, followed by cleavage to generate the aldehydic macrocycle, followed by further chemistry, including Horner-Emmons type reactions, to generate molecules with a variety of substituents to replace the aldehyde.
  • SfA dihydroxylation of a sanglifehrin
  • cleavage to generate the aldehydic macrocycle
  • further chemistry including Horner-Emmons type reactions
  • the present invention provides macrocyclic esters and derivatives thereof according to formula (I) below, or a pharmaceutically acceptable salt thereof:
  • Ri represents alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyi, alkenylcycloalkenyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkenylaryl or alkenylheteroaryl any of which groups may optionally be substituted by monocylic aryl or monocyclic heteroaryl;
  • R 3 represents H or (CO) x alkyl
  • R 4 represents H or OH
  • n a single or double bond save that when n represents a double bond R 4 represents H ;
  • n represents a single or double bond save that when m represents a double bond R 5 represents H;
  • analogue means one analogue or more than one analogue.
  • analogue(s) refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).
  • sanglifehrin(s) refers to chemical compounds that are structurally similar to sanglifehrin A but which differ slightly in composition (as in the
  • sanglifehrin-like compounds discussed in WO97/02285 and WO98/07743, such as sanglifehrin B.
  • HCV Hepatitis C Virus
  • RNA RNA
  • enveloped virus in the viral family Flaviviridae.
  • HIV Human Immunodeficiency Virus
  • the causative agent of Human Acquired Immune Deficiency Syndrome the causative agent of Human Acquired Immune Deficiency Syndrome.
  • bioavailability refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc. Various tests for bioavailability that would be familiar to a person of skill in the art are described herein (see also Egorin et al. 2002).
  • water solubility refers to solubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH 7.4, or in 5% glucose solution. Tests for water solubility are given below in the Examples as “water solubility assay”.
  • PBS phosphate buffered saline
  • the pharmaceutically acceptable salts of compounds of the invention include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like.
  • alkyl represents a straight chain or branched alkyl group, containing typically 1 -10 carbon atoms, for example a Ci -6 alkyl group.
  • Alkenyl refers to an alkyl group containing two or more carbons (for example 2-10 carbons e.g. 2-6 carbons) which is unsaturated with one or more double bonds.
  • alkyl groups examples include C 1 -4 alkyl groups such as methyl, ethyl, n-propyl, i-propyl, and n-butyl.
  • cycloalkyl represents a cyclic alkyl group, containing typically 3- 10 carbon atoms, optionally branched, for example cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. A branched example is 2-methylcyclopentyl.
  • Cycloalkenyl refers to a cyclic alkenyl group containing typically 5-10 carbon atoms, for example cyclopentyl, cyclohexenyl or cycloheptenyl. Cycloalkyl and cycloalkenyl groups may for example be monocyclic or bicyclic (including spirocyclic) but are suitably monocyclic.
  • cycloalkyl represents a cyclic alkyl group, containing typically 3- 10 carbon atoms, optionally branched, for example cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. A branched example is 2-methylcyclopentyl.
  • Cycloalkenyl refers to a cyclic alkenyl group containing typically 5-10 carbon atoms, for example cyclopentyl, cyclohexenyl or
  • Cycloalkyl and cycloalkenyl groups may for example be monocyclic or bicyclic (including spirocyclic) but are suitably monocyclic.
  • heterocyclyl represents a cycloalkyl group in which one or more one or more ring carbon atoms (e.g. 1 , 2 or 3 ring carbon atoms such as 1 or 2 e.g. 1 ) are replaced by heteroatoms selected from O, N and S.
  • ring carbon atoms e.g. 1 , 2 or 3 ring carbon atoms such as 1 or 2 e.g. 1
  • heteroatoms selected from O, N and S. Examples include morpholinyl, piperidinyl, pyrrolidinyl, piperazinyl and N-methyl piperazinyl.
  • heterocyclenyl represents a cycloalkenyl group in which one or more one or more ring carbon atoms (e.g. 1 , 2 or 3 ring carbon atoms such as 1 or 2 e.g. 1 ) are replaced by heteroatoms selected from O, N and S.
  • aryl groups include (except where indicated) monocyclic groups i.e. phenyl and bicyclic rings (e.g. 9 and 10 membered rings) which are aromatic or (in the case of bicyclic rings contain at least one aromatic ring).
  • a bicyclic ring may be fully aromatic e.g. naphthyl or may be partially aromatic (e.g. containing one aromatic ring), such as tetraline, indene or indane.
  • Preferred aryl is phenyl.
  • Aryl groups may optionally be substituted e.g. with one or more (e.g. 1 , 2 or 3) substituents e.g.
  • alkyl eg Ci -4 alkyl
  • hydroxyl e.g. Ci -4 alkyl
  • CF 3 e.g. CF 3
  • halogen e.g. Ci -4 alkoxy
  • nitro e.g. Ci -4 alkoxy
  • nitro e.g. Ci -4 alkoxy
  • nitro e.g. Ci -4 alkoxy
  • nitro e.g. Ci -4 alkoxy
  • nitro -S0 2 Me
  • cyano cyano and -CONH 2 .
  • heteroaryl groups include (except where indicated) monocyclic groups (e.g. 5 and 6 membered rings) and bicyclic rings (e.g. 9 and 10 membered rings) which are aromatic or (in the case of bicyclic rings contain at least one aromatic ring) and contain one or more heteroatoms (e.g. 1 , 2, 3 or 4) heteroatoms selected from N, O and S.
  • monocyclic groups e.g. 5 and 6 membered rings
  • bicyclic rings e.g. 9 and 10 membered rings
  • heteroatoms e.g. 1 , 2, 3 or 4
  • heteroatoms e.g. 1 , 2, 3 or 4
  • 5 membered heteroaryl rings include pyrrole, furan, thiophene, oxazole, oxadiazole, thiazole and triazole.
  • 6 membered heteroaryl rings include pyridine, pyrimidine and pyrazine.
  • bicyclic rings examples include fully aromatic rings such as quinoline, quinazoline, isoquinoline, indole, cinnoline, benzthiazole, benzimidazole, purine and quinoxaline and partially aromatic rings such as chromene, chromane, tetrahydroquinoline, dihydroquinoline, isoindoline and indoline.
  • Monocyclic heteroaryl groups are preferred. The aforementioned heteroaryl groups may be optionally substituted as described above for aryl groups.
  • connection to the remainder of the molecule may be through the aromatic portion or through the non-aromatic portion.
  • treatment includes prophylactic as well as therapeutic treatment.
  • Figure 1 A: HPLC Profile of Harvest Whole Broth Sample of sanglifehrin A, 5 &
  • Figure 6 Synthesised DNA fragment containing a region of homology upstream of the reductive loop of sanglifehrin module 12 (SEQ ID NO: 1 ).
  • Figure 7 MGo013 + MGo14 PCR product with inserted G at position1978 (SEQ ID NO: 4).
  • the present invention provides sanglifehrin macrocylic ester analogues, as set out above, methods for preparation of these compounds and methods for the use of these compounds in medicine.
  • the compound is a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl groups and methanol. In another embodiment it is not.
  • the variable p suitably represents 0 or 1. In one embodiment p represents 0 in another embodiment p represents 1 . In another embodiment p represents 2.
  • Ri represents -alkenylaryl
  • an example includes C 2-3 alkenylaryl e.g. - ethenylphenyl.
  • Ri represents -alkenylheteroaryl
  • an example includes C 2-3 alkenylheteroaryl e.g. -ethenylpyridinyl.
  • R-i represents alkyl, alkenyl, cycloalkyi, cycloalkenyl, alkylcycloalkyi, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, aryl, heteroaryl, alkylaryl,
  • R-i represents C 4- io alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkenylaryl or alkenylheteroaryl any of which groups may optionally be substituted by monocylic aryl or monocyclic heteroaryl;
  • R-i represents aryl or heteroaryl substituted by monocyclic aryl or monocyclic heteroaryl.
  • R-i may, for example, represent 4-biphenylyl in which either of the phenyl rings is optionally substituted.
  • N If -CH 3 is replaced by N, the group formed is -NH 2 -. If -CH 2 - is replaced by N, the group formed is -NH-. If -CHR- is replaced by N the group formed is -NR-.
  • nitrogen atoms within Ri may be primary, secondary or tertiary nitrogen atoms.
  • a carbon atom of Ri is replaced by a heteroatom, it is suitably replaced by O or N, especially O.
  • a carbon atom of Ri is replaced by a heteroatom such that Ri represents heterocyclyl, heterocyclenyl, alkylheterocyclyl, alkylheterocyclenyl, alkenylheterocyclyl or alkenylheterocyclenyl.
  • R-i contains more than one heteroatom, these should typically be separated by two or more carbon atoms.
  • R-i contains a heteroatom
  • the heteroatom is not positioned adjacent to the O atom to which R-i is attached.
  • R-i does not contain any heteroatom.
  • carbonyl When a carbon atom of Ri is replaced by a carbonyl, the carbonyl is suitably located adjacent to another carbon atom or a nitrogen atom. Suitably carbonyl groups are not located adjacent to sulphur or oxygen atoms.
  • Ri may represent -COCi -3 alkyl e.g. -COMe.
  • a carbon atom of Ri is not replaced by a carbonyl.
  • R-i may represent aryl or heteroaryl optionally substituted by monocyclic aryl or monocyclic heteroaryl, or -C 2-4 alkenyl.
  • R-i represents alkyl suitably it represents C 4- i 0 alkyl (e.g. C 4-6 alkyl).
  • Ri is selected from C 2- io alkyl (e.g. C 2-6 alkyl such as C 4 -6 alkyl), C 2- io alkenyl (e.g. C 2-6 alkenyl such as C 4-6 alkenyl) and aryl.
  • R-i is selected from C 4-6 alkyl, C 2-6 alkenyl and aryl.
  • exemplary halogen atoms are F, CI and Br, especially F and CI particularly F.
  • Ri moieties may be substituted by up to 6 halogen atoms (e.g. F atoms) for example up to 3 halogen atoms (e.g. F atoms).
  • halogen atoms e.g. F atoms
  • 3 halogen atoms e.g. F atoms
  • An exemplary halogenated Ri moiety is -CF 3 .
  • Ri represents alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyi, alkenylcycloalkenyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkenylaryl or alkenylheteroaryl any of which groups may optionally be substituted by monocylic aryl or monocyclic heteroaryl;
  • R-i does not represent methyl or -CHMe 2 .
  • R-i groups include those aryl or heteroaryl groups just mentioned in which the aryl or heteroaryl group is substituted.
  • R-i groups include cyclohexyl and methylcyclopentyl.
  • x represents 0.
  • Suitable R 3 represents H or (CO) x Ci -4 alkyl e.g. H or Ci -4 alkyl such as H or methyl, especially H.
  • n represents bond
  • m represents bond
  • R4 represents OH.
  • R3 represents H
  • R 4 represents OH
  • n represents bond
  • m represents bond
  • R 5 0 as represented by the following structure:
  • R-i represents C(CH 3 ) 3
  • R 3 represents H
  • R 4 represents OH
  • n represents bond
  • m represents bond
  • R 5 0 as represented by the following structure:
  • R-i represents C(CH 3 ) 3
  • R 3 represents H
  • R 4 represents H
  • n represents a double bond
  • m represents bond
  • R 5 0 as represented by the following structure:
  • R3 represents H
  • R 4 represents H
  • n represents bond
  • m represents bond
  • R 5 0 as represented by the following structure:
  • R-i represents phenyl
  • R 3 represents H
  • R 4 represents H
  • n represents bond
  • m represents bond
  • R 5 0 as represented by the following structure:
  • R 3 represents H
  • R 4 represents OH
  • n represents a single bond
  • m represents a single bond
  • R 5 0 as represented by the following structure:
  • the double bond at the C26,27 position may be in the cis form instead of the trans form.
  • the double bond at the C26,27 position is in the cis form, as represented by the following formula:
  • Such compounds may be produced during chemical synthesis.
  • the compounds of the invention are prepared by semi-synthetic derivatisation of a sanglifehrin.
  • Sanglifehrins may be prepared using methods described in WO97/02285 and WO98/07743, which documents are incorporated in their entirety, or additional methods described herein.
  • Sanglifehrins have also been produced by complex total synthetic chemistry which is capable of producing low amounts of material following extensive laboratory work. Semisynthetic methods for generating the sanglifehrin macrocylic aldehyde are described in US6, 124,453, Metternich et al., 1999, Banteli et al., 2001 and Sedrani et al., 2003.
  • a process for preparing certain compounds of formula (I) or a pharmaceutically acceptable salt thereof comprises:
  • R 8 groups which may be the same or different, independently represent alkyl (e.g. C 4 alkyl) or benzyl.
  • a process for preparing compounds of the invention comprises reacting a compound of formula II with an aldehydic macrocycle (compound of formula III).
  • a sanglifehrin such as SfA
  • modified Sharpless conditions catalytic osmium tetroxide
  • the use of the chiral ligands aids in promoting selectivity.
  • the resultant diol can then be cleaved oxidatively, using for instance sodium periodate.
  • the resultant compound of formula III can then be used as a substrate for derivatisation to an homologated ester.
  • a compound of formula II is dissolved in an aprotic solvent, cooled and the treated with a base, for example sodium hydride.
  • a compound of formula III is then added and the reaction warmed in temperature. After a suitable period of time the reaction is stopped and the compound of formula I is purified by standard conditions (e.g. preparative HPLC, preparative TLC etc).
  • Compounds of formula II may be known or readily synthesised from available alcohols (R 1 OH). As shown in scheme 1 (below) the alcohol may be used to treat chloroacetyl chloride or similar to form an alpha-chloroester. The alpha-chloroester is then treated in an Arbuzov reaction to generate the compound of formula II. Other routes to compounds of formula II will be apparent to one skilled in the art.
  • the methanol adduct may be prepared by fermentation and isolation from broth, or may be prepared from sanglifehrin A (WO97/02285).
  • a sanglifehrin macrocycle of the invention may be administered alone or in combination with other therapeutic agents.
  • Co-administration of two (or more) agents allows for lower doses of each to be used, thereby reducing side effect, can lead to improved potency and therefore higher SVR, and a reduction in resistance.
  • the sanglifehrin macrocycle of the invention is coadministered with one or more therapeutic agent s for the treatment of HCV infection, taken from the standard of care treatments.
  • a sanglifehrin macrocycle of the invention is co-administered with one or more other anti-viral agents, such as a STAT-C (specifically targeted agent for treatment of HCV), which could be one or more of the following: Non-nucleoside Polymerase inhibitors (e.g. ABT-333, ABT-072, BMS 791325, IDX375, VCH-222, Bl 207127, ANA598, VCH- 916, GS 9190, PF-00868554 (Filibuvir) or VX-759), Nucleoside or nucleotide polymerase inhibitors (e.g.
  • Non-nucleoside Polymerase inhibitors e.g. ABT-333, ABT-072, BMS 791325, IDX375, VCH-222, Bl 207127, ANA598, VCH- 916, GS 9190, PF-00868554 (Filibuvir) or VX-759
  • SCH503034 (Boceprevir), TMC435350, MK-7009 (Vaneprivir), R7227/ITMN-191 , EA-058, EA-063 or VX 985), NS5A inhibitors (e.g. A-831 , BMS 790052, BMS 824393, CY-102 or PPI-461 ), silymarin, NS4b inhibitors, serine C-palmitoyltransferase inhibitors, Nitazoxanide or viral entry inhibitors (e.g. PRO 206).
  • NS5A inhibitors e.g. A-831 , BMS 790052, BMS 824393, CY-102 or PPI-461
  • silymarin NS4b inhibitors
  • serine C-palmitoyltransferase inhibitors e.g. PRO 206.
  • a sanglifehrin macrocycle of the invention is co-administered with one or more other anti-viral agents (such as highly active antiretroviral therapy (HAART)) for the treatment of HIV, which could be one or more of the following: nucleoside reverse transcriptase inhibitors (NRTI) (e.g. Emtricitabine or Tenofovir), non-nucleoside reverse transcriptase inhibitors (NNRTI) (e.g. Rilipivirine or Efavirenz), protease inhibitors (PI) (e.g. Ritonavir or Lopinavir), fusion inhibitors (e.g. Maraviroc or Enfuvirtide), CCR5 inhibitors (e.g. Aplaviroc or Vicriviroc), maturation inhibitors (e.g. Bevirimat), CD4 monoclonal antibodies (e.g. Ibalizumab) and integrase inhibitors (e.g. Eltiegravir).
  • NRTI nucleoside reverse
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release
  • HPMC hydroxypropylmethylcellulose
  • HPC hydroxy-propylmethylcellulose
  • HPC hydroxy-propylcellulose
  • sucrose gelatin and acacia
  • lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example,
  • hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • agents such as preservatives and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • the dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.
  • the dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration.
  • the appropriate dosage can be readily determined by a person skilled in the art.
  • compositions may contain from 0.1 % by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.
  • the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.
  • RNA virus infections such as HCV or HIV infection or as an
  • -A compound according to the invention for use as a pharmaceutical for the treatment of muscular dystrophy;
  • composition comprising a compound according to the invention together with a pharmaceutically acceptable diluent or carrier;
  • composition comprising a compound according to the invention together with a pharmaceutically acceptable diluent or carrier further comprising a second or subsequent active ingredient, especially an active ingredient indicated for the treatment of viral infections such as HCV or HIV infection or as an immunosuppressant or an anti-inflammatory agent;
  • RNA virus infections such as HCV or HIV infection or muscular dystrophy
  • administering to a subject a therapeutically effective amount of a compound according to the invention
  • -Use of a compound according to the invention for the manufacture of a medicament for the treatment of viral infections such as HCV or HIV infection or as an immunosuppressant or an anti-inflammatory agent.
  • the sanglifehrin producer Streptomyces sp. A92-3081 10 (DSM no 9954, purchased from DSMZ, Braunschweig, Germany) also termed BIOT-4253 and BIOT-4370 is maintained on medium oatmeal agar, MAM, or ISP2 (see below) at 28 °C.
  • Streptomyces sp. A92-3081 10 was grown on oatmeal agar at 28 °C for 7-10 days. Spores from the surface of the agar plate were collected into 20% w/v sterile glycerol in distilled and storec in 0.5-ml aliquots at -80 °C. Frozen spore stock was used for inoculating seed media SGS or SM25- 3. The inoculated seed medium was incubated with shaking between 200 and 300 rpm at 5.0 or 2.1 cm throw at 27 °C for 24 hours.
  • the fermentation medium SGP-2 or BT6 were inoculated with 2.5%- 10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 err throw at 24 °C for 4-5 days. The culture was then harvested for extraction.
  • Glycerol (Fisher scientific, G/0650/25) 7.50 g yeast extract (Becton Dickinson, 212770) 1.35 g malt extract (Becton Dickinson, 218630) 3.75 g potato starch (soluble) (Signma, S2004) 7.50 g
  • NZ-amine A (Sigma, C0626) 2.50 g toasted soy flour, Nutrisoy (ADM, 063-160) 2.50 g
  • Solvent A is Water + 0.1 % Formic Acid
  • Solvent B is Acetonitrile + 0.1 % Formic Acid
  • LCMS is performed on an integrated Agilent HP1 100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive ion mode using the chromatography and solvents described above.
  • Solvent A is Water + 0.1 % Formic Acid
  • Solvent B is Acetonitrile + 0.1 % Formic Acid MS conditions
  • MS operates in switching mode (switching between positive and negative), scanning from 150 to 1500 amu.
  • Solvent gradient A1 (e.g. for cpd 1 ):
  • Solvent gradient A2 (e.g. for cpd 5):
  • Solvent A is H 2 O-0.025% FA- 1 mM NH 4 OAC
  • Solvent gradient A3 (e.g. for cpds 11 and 12):
  • Solvent B is MeOH-0.025% FA- 1 mM NH 4 OAC
  • Solvent gradient A4 (e.g. for compound 12):
  • Solvent A is H 2 O-0.025% FA- 1 mM NH 4 OAC
  • Solvent B is MeOH-0.025% FA- 1 mM NH 4 OAC negative scan mode
  • Antiviral efficacy against genotype 1 HCV may be tested as follows: One day before addition of the test article, Huh5.2 cells, containing the HCV genotype 1 b l389luc-ubi-neo/NS3- 375.1 replicon (Vrolijk et al., 2003) and subcultured in cell growth medium [DMEM (Cat No.
  • microtitre plates are incubated overnight (37°C, 5% C0 2 , 95-99% relative humidity), yielding a non-confluent cell monolayer. Dilution series are prepared; each dilution series is performed in at least duplicate. Following assay setup, the microtitre plates are incubated for 72 hours (37°C, 5% C0 2 , 95-99% relative humidity).
  • the assay medium is aspirated, replaced with 75 ⁇ _ of a 5% MTS (Promega) solution in phenol red-free medium and incubated for 1 .5 hours (37°C, 5% C0 2 , 95-99% relative humidity). Absorbance is measured at a wavelength of 498nm (Safire 2 , Tecan) and optical densities (OD values) are converted to percentage of untreated controls.
  • assay medium is aspirated and the cell monolayers are washed with PBS.
  • the wash buffer is aspirated, 25 ⁇ _ of Glo Lysis Buffer (Cat. N°. E2661 , Promega) is added after which lysis is allowed to proceed for 5min at room temperature.
  • 50 ⁇ _ of Luciferase Assay System (Cat. N°. E1501 , Promega) is added and the luciferase luminescence signal is quantified immediately (1000ms integration time/well, Safire 2 , Tecan). Relative luminescence units are converted to percentage of untreated controls.
  • the EC50 and EC90 represent the concentrations at which respectively 50% and 90% inhibition of viral replication would be observed.
  • the CC50 value derived from the dose-response curve
  • a concentration of compound is considered to elicit a genuine antiviral effect in the HCV replicon system when, at that particular concentration, the anti-replicon effect is above the 70% threshold and no more than 30% reduction in metabolic activity is observed.
  • the replicon cells (subgenomic replicons of genotype 1 a (H77) and 2a (JFH-1 )) were grown in Dulbecco's modified essential media (DMEM), 10% fetal bovine serum (FBS), 1 % penicillin-streptomycin (pen-strep), 1 % glutamine, 1 % non-essential amino acids, 250 ⁇ g ml G418 in a 5% C0 2 incubator at 37°C. All cell culture reagents were purchased from Mediatech (Herndon, VA).
  • the replicon cells were trypsinized and seeded at 5 x 10 3 cells per well in 96-well plates with the above media without G418. On the following day, the culture medium was replaced with DMEM containing compounds serially diluted in the presence of 5% FBS.
  • the HCV replicon antiviral assay examines the effects of compounds in a serial of compound dilutions. Briefly, the cells containing the HCV replicon were seeded into 96-well plates. Test article was serially diluted with DMEM plus 5% FBS. The diluted compound was applied to appropriate wells in the plate. After 72 hr incubation at 37°C, the cells were processed.
  • the intracellular RNA from each well was extracted with an RNeasy 96 kit (Qiagen).
  • the level of HCV RNA was determined by a reverse transcriptase-real time PCR assay using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA) and an ABI Prism 7900 sequence detection system (Applied Biosystems) a as described previously (Vrolijk et al., 2003).
  • the cytotoxic effects were measured with TaqMan® Ribosomal RNA Control Reagents (Applied Biosystems) as an indication of cell numbers.
  • the amount of the HCV RNA and ribosomal RNA were then used to derive applicable IC50 values (concentration inhibiting on replicon replication by 50%).
  • Water solubility may be tested as follows: A 10 mM stock solution of the sanglifehrin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above.
  • solubility in PBS at pH7.4 may be tested as follows: A calibration curve is generated by diluting the test compounds and control compounds to 40 ⁇ , 16 ⁇ , 4 ⁇ , 1 .6 ⁇ , 0.4 ⁇ , 0.16 ⁇ , 0.04 ⁇ and 0.002 ⁇ , with 50% MeOH in ⁇ 2 0. The standard points are then further diluted 1 :20 in MeOH:PBS 1 :1 . The final concentrations after 1 :20 dilution are 2000nM, 800nM, 200nM, 80nM, 20nM, 8nM, 2nM and 1 nM. Standards are then mixed with the same volume (1 :1 ) of ACN containing internal standard (hydroxymacrocycle, 6). The samples are centrifuged (5min, 12000rpm), then analysed by LC/MS.
  • Test compounds are prepared as stock solutions in DMSO at 10mM concentration.
  • the stock solutions are diluted in duplicate into PBS, pH7.4 in 1 .5ml_ Eppendorf tubes to a target concentration of 100 ⁇ with a final DMSO concentration of 1 % (e.g. 4 ⁇ _ of 10mM DMSO stock solution into 396 ⁇ _ 100mM phosphate buffer).
  • Sample tubes are then gently shaken for 4 hours at room temperature. Samples are centrifuged (10min, 15000rpm) to precipitate undissolved particles. Supernatants are transferred into new tubes and diluted (the dilution factor for the individual test article is confirmed by the signal level of the compound on the applied analytical instrument) with PBS. Diluted samples are then mixed with the same volume (1 :1 ) of MeOH. Samples are finally mixed with the same volume (1 :1 ) of ACN containing internal standard (hydroxymacrocycle, 6) for LC-MS/MS analysis.
  • Cell permeability may be tested as follows: The test compound is dissolved to 10mM in DMSO and then diluted further in buffer to produce a final 10 ⁇ dosing concentration. The fluorescence marker lucifer yellow is also included to monitor membrane integrity. Test compound is then applied to the apical surface of Caco-2 cell monolayers and compound permeation into the basolateral compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS is used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol).
  • In vivo assays may also be used to measure the bioavailability of a compound.
  • a compound is administered to a test animal (e.g. mouse or rat) both intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time.
  • the time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below.
  • mice are dosed with 1 , 10, or 100 mg/kg of the compound of the invention or the parent compound i.v. or p.o.. Blood samples are taken at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420 and 2880 minutes and the concentration of the compound of the invention or parent compound in the sample is determined via HPLC.
  • the time-course of plasma concentrations can then be used to derive key parameters such as the area under the plasma concentration- time curve (AUC - which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma drug concentration, the time at which maximum plasma drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half life, total body clearance, steady-state volume of distribution and F%. These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Egorin et al. 2002, and references therein.
  • the concentration of the compound of the invention or parent compound in the sample was determined via LCMS as follows:20 ⁇ _ of blood:H 2 0 (1 :1 , v/v)/PK sample was added with 20 ⁇ _ Internal standard (hydroxyl macrocycle, 6) at 100 ng/mL, 20 ⁇ _ working solution/MeOH and 150 ⁇ _ of ACN, vortexed for 1 minute at 1500 rpm, and centrifuged at 12000 rpm for 5 min. The supernatant was then injected into LC-MS/MS. The time-course of blood concentrations was plotted and used to derive area under the whole blood concentration-time curve (AUC - which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation). These values were used to generate the oral bioavailability (F%) and other PK parameters where possible.
  • Huh-7 and HepG2 cells obtained from ATCC were grown in Dulbecco's modified essential media (DMEM) containing 10% fetal bovine serum (FBS), 1 % penicillin-streptomycin (pen-strep) and 1 % glutamine; whereas CEM cells (human T-cell leukemia cells obtained from ATCC) were grown in RPMI 1640 medium with 10% FBS, 1 % pen-strep and 1 % glutamine.
  • DMEM Dulbecco's modified essential media
  • FBS fetal bovine serum
  • pen-strep penicillin-streptomycin
  • glutamine human T-cell leukemia cells obtained from ATCC
  • PBMCs Banded PBMCs were gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation. After the final wash, cells were counted by trypan blue exclusion and resuspended at 1 x 10 7 cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 ⁇ g/mL PHA-P. The cells were allowed to incubate for 48-72 hours at 37°C.
  • FBS Fetal Bovine Serum
  • PBMCs were centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 10 ⁇ g/mL gentamycin, and 20 U/mL recombinant human IL-2.
  • Compound cytotoxicity was evaluated by testing half-log concentrations of each compound in triplicate against the cells described above.
  • Cell containing medium alone served as the cell control (CC).
  • Huh-7 and HepG2 cells were seeded in 96-well plates at a
  • PBMCs were diluted in fresh medium and plated in the interior wells of a 96 well round bottom microplate at 5 x 10 4 cells/well in a volume of 100 L. Similarly, CEM cells were plated at 1 x 10 4 cells/well. Then, 100 ⁇ of 2X preparations of the test drugs were added in appropriate wells in a standard format. The cultures were maintained for six to seven days and then processed for cytotoxicity determination.
  • Cytotoxicity was determined using CytoTox-ONETM homogeneous membrane integrity assay kit (Promega). The assay measures the release of lactate dehyrodgenase (LDH) from cells with damaged membranes in a fluorometric, homogeneous format. LDH released into the culture medium is measured with a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of lysed cells. Six serially diluted concentrations of each compound were applied to the cells to derive where applicable TC50 (toxic concentration of the drug decreasing cell viability by 50%) and TC90 (toxic concentration of the drug decreasing cell viability by 90%) values.
  • TC50 toxic concentration of the drug decreasing cell viability by 50%
  • TC90 toxic concentration of the drug decreasing cell viability by 90%
  • an in vitro ATPase assay from Solvo Biotechnology Inc. can be used (Glavinas et al., 2003).
  • the compounds (at 0.1 , 1 , 10 and 100 ⁇ ) are incubated with MDR1 or MRP2 membrane vesicles both in the absence and presence of vanadate to study the potential ATPase activation.
  • similar incubations are conducted in the presence of
  • ATPase activity is measured by quantifying inorganic phosphate spectrophotometrically.
  • Activation is calculated from the vanadate sensitive increase in ATPase activity. Inhibition is determined by decrease in verapamil/sulfasalazine mediated ATPase activity.
  • PBMC Peripheral blood mononuclear cell
  • Culture conditions included: cell populations A & B alone and a mixed population of cells A&B in the absence or presence of test compounds, each at 6 different concentrations. Compounds were tested at doses ranging from 10 ⁇ to 0.0001 ⁇ in 1 -log increments. Control wells contained a comparable concentration of vehicle (0.5% DMSO) to that present in the test compound wells. Cultures were established in triplicate in a 96 well plate and incubated at 37°C in 5% C0 2 in a humidified atmosphere. 3H-thymidine was added on day 6 after assay set up and harvested 24hrs later. The levels of proliferation between the different culture conditions were then compared.
  • each dilution of test compound to inhibit proliferation in the MLR was calculated as percentage inhibition. This allowed estimation of the IC 50 (concentration of test compound which resulted in a 50% reduction of counts per minute).
  • the X axis was transformed to a log scale. Non-linear regression was used to fit to the mean data points. A sigmoidal variable slope was selected.
  • Antiviral efficacy against HIV may be tested as follows: Blood derived CD4+ T- lymphocytes and macrophages are isolated as described previously (Bobardt et al., 2008). Briefly, human PBMCs were purified from fresh blood by banding on Ficoll-Hypaque (30 min, 800 g, 25°C). Primary human CD4+ T cells were purified from PBMCs by positive selection with anti-CD4 Dynabeads and subsequent release using Detachabead.
  • monocytes were purified from human PBMCs by negative selection and activated and cultured at a cell concentration of 106/ml in DMEM, supplemented with 10% FCS, MEM amino acids, L-glutamine, MEM vitamins, sodium pyruvate, and penicillin (100 units/ml), streptomycin (100 mg/ml), and 50 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and maintained at 37°C in a humidified atmosphere supplemented with 5% C0 2 .
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CD4+ HeLa cells, Jurkat cells, activated CD4+ peripheral blood T-lymphocytes and macrophages (500,000 cells/100 ⁇ _) were incubated with pNL4.3-GFP (X4 virus) or pNL4.3- BaL-GFP (R5 virus) (100 ng of p24) in the presence of increasing concentrations of test article, Forty-eight hours later, infection was scored by analyzing the percentage of GFP-positive cells by FACS and EC 50 calculated.
  • This assay was used to measure the disruption of Cyp-NS5A complexes, which can be used to show the potency of interaction with Cyclophilin D. Briefly, production and purification of recombinant GST, GST-CypD and Con1 NS5A-His proteins was carried out as described previously (Chatterji et al., 2010). Nunc MaxiSorb 8-well strip plates were coated with GST or GST-CypD for 16 h at 4 ° C and blocked.
  • NS5A-His (1 ng/mL) was added to wells in 50 ⁇ _ of binding buffer (20 mM Tris pH 7.9, 0.5 M NaCI, 10% glycerol, 10 mM DTT and 1 % NP-40) for 16 h at 4 ° C. Captured NS5A-His was subsequently detected using mouse anti-His antibodies (1 g/mL) (anti-6xHis, Clontech) and rabbit anti-mouse-horseradish peroxidase phosphatase (HRP) antibodies (1 :1000 dilution). All experiments were conducted twice using two different batches of recombinant CypD and NS5A proteins.
  • binding buffer 20 mM Tris pH 7.9, 0.5 M NaCI, 10% glycerol, 10 mM DTT and 1 % NP-40
  • CypD was equilibrated for 1 h at 5°C with selected test article using a drug concentration range from 0.1 to 20 nM. The reaction was started by addition of the peptide, and the change in absorbance was monitored spectrophotometrically at 10 data points per second. The blank rates of hydrolysis (in the absence of CypD) were subtracted from the rates in the presence of CypD. The initial rates of the enzymatic reaction were analyzed by first-order regression analysis of the time course of the change in absorbance.
  • Vegetative cultures were prepared by inoculating 0.2 mL from a spore stock of
  • the culture flasks were incubated at 27°C, 250 rpm (2.5 cm throw) for 24 h.
  • the whole broth (30 L) was clarified by centrifugation.
  • the resulting cell pellet was extracted twice with ethyl acetate (2 x 10 L), each by stirring for 1 hour with overhead paddle stirrer and leaving to settle before pumping off solvent.
  • the ethyl acetate layers were then combined (-20 L) and the solvent removed under reduced pressure at 40°C to obtain an oily residue.
  • This oily residue was then suspended in 80:20 methanol :water (total volume of 500 mL), and twice extracted with hexane (2 x 500 mL).
  • the 80:20 methanokwater fraction was then dried under reduced pressure to yield a crude dry extract which contained SfA and SfB.
  • This extract was dissolved in methanol (100 ml), mixed with 15 g Silica gel and dried to a powder.
  • the powder was loaded into a silica gel column (5 x 20 cm) packed in 100% CHCI 3 .
  • the methanol concentration was increased stepwise by 1 % and 250 ml fractions collected.
  • the methanol concentration was increased stepwise by 2% up to 8%.
  • Example 8 Biological data - In vitro evaluation of HCV antiviral activity in the replicon system Compounds were analysed in the replicon assay as described in the General Methods. Cyclosporine A, 1 , sanglifehrin A, 5, and the hydroxymacrocycle, 6 were included as a comparison.
  • 10, 11 and 12 are all very potent in the Huh5.2 replicon assay (as shown by the low EC50), with good selectivity against the cell line (as shown by a high selectivity index).
  • the previously described macrocylic sanglifehrin hydroxymacrocycle, 6, is much less potent at HCV inhibition, and cyclosporine A, 1 and sanglifehrin A, 5 are less potent and both have poorer selectivity indices.
  • Solubility of the compounds in PBS pH 7.4 was analysed as described in the General Methods. Cyclosporine A, 1 and sanglifehrin A, 5 were included as a comparison.
  • the compounds of the invention, 10, 11 and 12 all have increased solubility when compared to sanglifehrin A (5) and some improvement over cyclosporine A (1 ).
  • 12 is more potent in the 1 a and 2a replicon assays than Sanglifehrin A (as shown by the low EC50), and shows activity across genotypes.
  • the CC50 for Sanglifehrin A and compound 12 was >500nM, showing that the activity of 12 was selective.
  • 12 has improved oral bioavailability compared to compound 11 and
  • sanglifehrin A whilst compound 11 has improved oral bioavailability compared to sanglifehrin A.
  • Cytotoxicity of compounds was evaluated against in the hepatocyte cell lines Huh7 and HepG2, human PBMCs and the cancer cell line CEM, as described in the general methods. Sanglifehrin A, 5, was included as a comparison. The maximum concentration tested was 50 ⁇ .
  • 12 is >10 fold more potent at inhibition of antiCD3/antiCD28 stimulated cell proliferation than the natural compound sanglifehrin A, 5.
  • the compound of the invention shows potent disruption of the CypD-NS5A complex, at a more potent level than CsA, 1. It was also confirmed that these assays gave comparable data (and similar rank orders) to a PPIase assay measuring direct inhibition of CypD isomerase activity (data not shown - see general methods for details of methodology).
  • reaction was stopped by the addition of water (2 mL) and the resultant aqueous mixture extracted with ethyl actetate (5 x 4 mL). The combined organics were washed with water (1 x 20 mL), dried with a small portion of Na 2 S0 4 , and removed in vacuo.
  • Example 16 Generation of bio-engineered Streptomyces sp. A92-3081 10 (DSM9954) (BIOT- 4370) strains in which the reductive loop of module 12 of the biosynthetic cluster for sanglifehrin biosynthesis is replaced by the reductive loop from rapamycin module 13 or sanglifehrin module 6 using a reductive loop swap strategy.
  • the reductive loop of sanglifehrin module 12 contains a ketoreductase which is responsible for the hydroxyl group at C17 of the sanglifehrin molecule.
  • the reductive loops from both rapamycin module 13 and sanglifehrin module 6 contain all of the functional domains to result in full processing of the beta-keto group to result in a methylene; specifically they contain a keto reductase to reduce the keto to a hydroxyl group, a dehydratase to remove water and result in a double bond, and an enoyl reductase to reduce the double bond to a methylene.
  • Positions of DNA fragments used in this example are given according to the sequence available in January 201 1 but reported as approximate because Genbank DNA sequences can be updated.
  • This 2072 bp DNA fragment (SEQ ID NO: 1 ) shown in Figure 6 contains a region of homology upstream of the reductive loop of sanglifehrin module 12 (approximately from 86654 bp - 88798 bp in the published sequence Genbank accession number FJ809786.1 ) along with additional sequences both 5' and 3' to incorporate restriction enzyme sequences to aid cloning.
  • This fragment (SEQ ID NO:1 ) was synthesised by GenScript (860 Centennial Ave., Piscataway, NJ 08854, USA) and provided, according to the GenScript protocol with 12 protective flanking bases on each side which do not participate in the cloning beyond this point, in pUC57 resulting in plasmid pMGo128.
  • Oligos MGo013 SEQ ID NO: 2 and MGo014 (SEQ ID NO: 3) were used to amplify a 1994 bp DNA fragment (SEQ ID NO: 4) in a standard PCR reaction using cosmid pTL3102 (Qu et al. 201 1 ) DNA as the template and KOD Hot Start DNA polymerase.
  • a 5' extension was designed in each oligo to introduce restriction sites to facilitate cloning of the amplified fragment.
  • genomic DNA from Streptomyces sp. A92-3081 10 (DSM9954) (BIOT-4370) could have been used as the template for this PCR reaction to give the same DNA fragment, or the DNA fragment could be obtained by DNA synthesis for example using GenScript (860
  • the resulting 1995 bp PCR product (SEQ ID NO: 4) contains a region of homology downstream of the reductive loop of sanglifehrin module 12 (approximately from 90415 bp - 92381 bp in the published sequence genbank accession number FJ809786.1 ) with an undesired insertion, G at position 1978 (see Figure 7; inserted G is bold and underlined).
  • the 1995 bp PCR product (SEQ ID NO: 4) was cloned into pUC19 (New England Biolabs) that had been linearised with Sma ⁇ and dephosphorylated, resulting in plasmid pMGo123.
  • the orientation of the 1995 bp PCR product (SEQ ID NO: 4) in pUC19 was such that the Hind ⁇ site on the insert was adjacent to the Hind ⁇ site of the pUC19 polylinker.
  • the sequence of the insert in pMGo123 was confirmed by sequencing.
  • the upstream and downstream regions of homology of the sanglifehrin reductive loop of module 12 are cloned together as follows: The 2065 bp upstream region is excised from pMGo128 by digestion with EcoRI and Xho ⁇ and the 1944 bp downstream region is excised from pMGo125 by digestion with Xho ⁇ and Hind ⁇ . Both fragments are cloned together into the large backbone fragment generated when pUC19 (New England Biolabs) is digested with EcoRI and Hind ⁇ in a three part ligation. Plasmids containing both inserts correctly cloned are identified by restriction enzyme analysis, one correct plasmid is designated pMGo130.
  • pMGo130 is designed such that a reductive loop on a suitable NheUBglW fragment, can be cloned into the Nhe ⁇ and BglW sites to yield a portion of a type I PKS module in which the DNA sequence is in frame and can be translated to give an amino acid sequence.
  • the exact positioning of these sites in the in-coming loop is crucial in maintaining the frame of the sequence and this translation into a functional amino acid sequence.
  • Rapamycin module 13 reductive loop has been used previously as a donor loop in other systems (eg. Gaisser et al., 2003). Rapamycin module 13 loop, flanked by appropriate regions of homology from avermectin module 2 is present in pPF137 (Gaisser et al., 2003). pPF137 is constructed from pJLK137 as described in Gaisser et al 2003. The full description of the construction of pJLK137 is contained within International patent application WO00/01827/1998 and references therein. A brief summary follows: The rapamycin module 13 loop was isolated by PCR amplification using the following oligos.
  • rapamycin cos 31 which contain introduced restriction enzyme sites, and using the template rapamycin cos 31 (Schwecke et al. 1995).
  • This fragment was cloned into pUC18 previously digested with Sma ⁇ and dephoshorylated to give pJLK120.
  • This loop was then introduced into pJLK133, which was constructed as follows: The linker was removed from pJLK1 17 on a Bgl ⁇ INhe ⁇ fragment and cloned between 2 regions of homology to avermectin module 2 to give pJLK133.
  • the rapamycin module 13 reductive loop was cloned from pJLK120 as a Bgl ⁇ /Nsi ⁇ fragment into Bgl ⁇ /Nsi ⁇ digested pJLK133.
  • pJLK1 17 (refer to International patent application WO00/01827/1998 and references therein) is an expression plasmid containing a PKS gene comprising the erythromycin loading module, the first and the second extension modules of the erythromycin PKS and the erythromycin chain terminating thioesterase, except the DNA segment between the end of the acyltransferase (AT) and the beginning of the acyl carrier protein (ACP) has been substituted by a synthetic oligonucleotide linker containing the recognition sites of the following restriction enzymes; ⁇ , BglW, Sna ⁇ , Pst ⁇ , Spe ⁇ , Nsi ⁇ , Bsu36 ⁇ , and Nhe ⁇ and was made in multiple steps as described in the patent application.
  • the first linker containing vector, pJLK1 14 contains the generated by annealing the oligos Plf (SEQ ID NO: 10) and Plb (SEQ ID NO: 1 1 ).
  • the plasmid pJLK1 17 was constructed by replacing the 5' end of the linker of pJLK1 14 with a fragment in which the only difference is that the Hpa ⁇ site, GTTAAC is replaced by an Nhe ⁇ site, GCTAGC.
  • the rapamycin module 13 loop could be amplified directly as a Bgl ⁇ /Nhe ⁇ fragment for example using the oligos SEQ ID NO: 8 as shown above and SEQ ID NO:12
  • rapamycin module 13 reductive loop was cloned from pPF137 into pKC1 139WMB02 as a Bgl ⁇ /N e ⁇ fragment to give pKC1 139WMB02-137.
  • pKC1 139WMB02 is a pKC1 139-based plasmid and contains a 7.8 kb DNA fragment containing the rapamycin module 1 1 reductive loop and flanking regions. It has been engineered such that the reductive loop can be excised as a Bgl ⁇ /Nhe ⁇ fragment and replaced with other loops.
  • pKC1 139WMB02-137 was constructed to effect a loop swap in rapamycin and contains the rapamycin module 13 reductive loop with flanking regions from rapamycin module 1 1.
  • rapamycin module 13 loop is cloned from pKC1 139WMB02-137 as a Bgl ⁇ /N e ⁇ fragment. This is the identical fragment that can be obtained from pPF137, or pJLK120 or by carrying out an equivalent PCR reaction using the oligo sequences provided and genomic DNA and cloning it into a suitable vector such as pUC18 or pUC19.
  • the sanglifehrin reductive loop of module 6 is obtained as follows: Oligos MGo019 (SEQ ID NO: 13) and MGo020 (SEQ ID NO: 14) are used to amplify a 3176 bp DNA fragment (SEQ ID NO: 15) in a standard PCR reaction using KOD Hot Start DNA polymerase and the 5 kb - 6 kb fraction of AlwN ⁇ digested genomic DNA from Streptomyces sp. A92-3081 10 (DSM9954) (BIOT-4370) as the template.
  • This fraction contains the 5402 bp AlwW fragment of the sanglifehrin gene cluster (approximately from 56578 bp - 61979 bp in the published sequence genbank accession number FJ809786.1 ).
  • undigested genomic DNA from Streptomyces sp. A92-3081 10 (DSM9954) (BIOT-4370) is used as the template.
  • Genomic DNA is obtained using the Edge BioSystems bacterial genomic DNA purification kit (Edge BioSystems, 201 Perry Parkway, Suite 5, Gaithersburg, MD 20877, USA).
  • a 5' extension is designed in each oligo to introduce restriction sites to facilitate cloning of the amplified fragment in-frame with the flanking regions.
  • the 3176 bp PCR product (SEQ ID NO: 15) contains the reductive loop of sanglifehrin module 6 (approximately from 57166 bp - 60326 bp in the published sequence genbank accession number FJ809786.1 ).
  • the 3176 bp PCR product (SEQ ID NO: 15) is cloned into pUC19 (New England Biolabs) that has been linearised with Sma ⁇ and dephosphorylated, resulting in plasmid pMGo127.
  • pKC1 139WMB02-137 and pMGo127 are each digested with Nhe ⁇ and BglW to isolate the rapamycin module 13 reductive loop and the sanglifehrin module 6 reductive loop.
  • Each loop is cloned into pMGo130 digested with Nhe ⁇ and BglW. Insert-containing plasmids are analysed by restriction enzyme analysis, one correct plasmid containing rapamycin module 13 reductive loop is designated pMGo132 and one correct plasmid containing sanglifehrin module 6 reductive loop is designated pMGo133.
  • 0.8 mL of each culture is used to inoculate 10 mL liquid 2TY containing apramycin (50 ⁇ g mL), kanamycin (25 ⁇ g mL) and chloramphenicol (12.5 ⁇ g mL) in a 50 mL Falcon tube and incubated at 37°C 250 rpm until OD 6 oonm ⁇ 0.5 is reached.
  • the resulting cultures are centrifuged at 3500 rpm for 10 min at 4°C, washed twice with 10 mL 2TY medium using centrifugation to pellet the cells after each wash.
  • the resulting pellets are resuspended in 0.5 mL 2TY and kept on ice ready for use. This process is timed to coincide with the completion of preparation of Streptomyces spores described below.
  • Spores of Streptomyces sp. A92-3081 10 are harvested from a 1 -2 week old confluent plate by resuspending in ⁇ 3 mL 20 % glycerol and splitting equally between 2 Eppendorf tubes. Alternatively, -1.5 mL of a cryopreserved spore suspension prepared in the same way is used. Spores are centrifuged (6000 rpm, 5 min room temperature) and washed twice with 1 mL 50 mM TES buffer before resuspending in 0.5 mL 50 mM TES buffer. This tube is heat shocked at 50°C for 10 min in a water bath before adding 0.5 mL of TSB medium and incubating in an Eppendorf Thermomixer compact at 37°C for 4-5 hours.
  • the prepared E. coli ET12567 pUZ8002 pMGo136 and £. coli ET12567 pUZ8002 pMGo137 are each mixed with BIOT-4370 at ratios 1 :1 (100 ⁇ each strain) and 1 :3 (100 ⁇ E. coli + 300 ⁇ - BIOT-4370) and immediately spread on R6 plates and transferred to a 37°C incubator. After approximately 2 hours incubation these plates are overlaid with 2 mL of sterile water containing nalidixic acid to give a final in-plate concentration of 50 ⁇ g L.
  • Plates are returned to the 37°C incubator overnight before overlaying with 2 mL of sterile water containing apramycin to give a final in-plate concentration of 20-25 ⁇ g L.
  • the plates are initially incubated for 16-18 hours, then overlaid with the nalidixic acid solution and allowed to dry for 1 -2 hours before being overlaid with the apramycin solution.
  • Ex-conjugant colonies appear after -4-7 days and are patched onto ISP4 media containing apramycin (25 ⁇ g L) and nalidixic acid (50 ⁇ g L) and incubated at 37°C.
  • Sporulated single colonies are doubly patched to ISP4 plates with and without apramycin (25 ⁇ g L) to identify colonies which loose the plasmid and allowed to grow -7 days before testing for production of sanglifehrins and sanglifehrin analogues.
  • Strains selected for analysis are those that do not grow in the presence of apramycin, indicating loss of the resistance marker desirably by secondary recombination.
  • a single -7 mm agar plug of each well sporulated patch is used to inoculate 7 mL of sterile SM25-3 media and incubated at 27°C 200 rpm in a 2 inch throw shaker. After 48 hours of growth 0.7 mL of each culture is transferred to a sterilised falcon tube containing 7 mL of SGP6 media (30 g/L Nutrisoy (Toasted Soy Flour), 60 g/L glycerol, 21 g/L MOPS; pH 6.8) with 5 % HP20 resin. Cultures are grown at 24°C 300 rpm on a 1 inch throw shaking incubator for 5 days before harvest.
  • Extracts of strains are analysed by HPLC. Strains that produced sanglifehrin A and B are not analysed further as this result indicates reversion to wild type. Strains lacking sanglifehrin A and B production and showing peaks consistent with the production of 17-deoxy-sanglifehrin A and 17-deoxy-sanglifehrin B are taken forward.
  • a strain producing 17-deoxy sanglifehrin A is then grown using a similar method to that described in Example 1 , the compound isolated using a similar method to that described in Example 2, and the aldehyde generated using a similar method to that described in example 3. This is then used as a template for semisynthesis as described to generate compounds of formula 1 .
  • Hepatitis C virus NS5A protein is a substrate for the Peptidyl-Prolyl cis/trans isomerase activity of Cyclophilins A and B. J Biol Chem. 284:13589-13601 Han, X., Yoon, S.H. et al., "Cyclosporin A and sanglifehrin A enhance chemotherapeutic effect of cisplatin in C6 glioma cells" Oncology Reports 23:1053-1062
  • Virus NS3-4A Protease or NS5B polymerase inhibitors enhance antiviral activity and suppress the emergence of resistance.”

Abstract

L'invention concerne entre autres des composés de formule (I) : destinés à être utilisés comme inhibiteurs et/ou immunosuppresseurs de la cyclophiline.
PCT/GB2011/050232 2010-02-09 2011-02-09 Composés à base de sangliféhrine WO2011098805A1 (fr)

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WO2012131377A1 (fr) * 2011-03-29 2012-10-04 Biotica Technology Limited Composés macrocycliques et leurs procédés de production
WO2013061052A1 (fr) 2011-10-24 2013-05-02 Biotica Technology Limited Nouvelle forme pharmaceutique
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